The focus of this proposal is on the mechanism of host lysis by bacteriophage. Building on recent progress, it is proposed to investigate the biochemical and genetic properties of holins, smallphage-encoded membrane proteins that act as the timers of phage infections. Holins have the remarkable ability to accumulate during the phage infective cycle without harming the cell, then suddenly triggering to permeabilize the membrane. This terminates the infection and activatesmuralytic enzymes called endolysins, or lysozymes, resulting in degradationof the cell wall, leading to bursting of the cell and release of the progeny virions. The work is aimed at determining how these proteins can form holes inmembranes, and how the scheduling of the hole-forming event is programmed into the sequence of the holin. Fundamental issues of lipid-protein and protein-protein interactions in membranes will be addressed, including an investigation of how integral membranedomains of some lysis proteins actually are able to exit the membrane upon physiological cues. The holin-endolysin mode is completely general for all phages except those with very small genomes. However, single-stranded DMA and RNAphages, limited to 3 - 10genes for their entire genome complement, accomplish host lysis by expressing single genes. In two of these cases, recent progress has shown that the phage lysis protein causes lysis by inhibiting different enzymes in the murein precursor biosynthetic pathway. It is proposed to investigate the molecular basis by which these "protein antibiotics" effect inhibition of these conserved enzymes. Other small single-stranded RNA phages effect lysis by an unknown mechanism, the elucidation of which is another goal of this project. Public health implications: These studies are critical to our understanding of how bacterial viruses, or phages, kill their prey and effect dispersal of their progeny. This may have direct practical benefits because there is a growing consensus that phages, as natural antibacterial agents, will become an important tool in combating bacterial pathogens, which are increasingly resistant to available antibiotics. In addition, the research may reveal new modes for design of chemical antibiotics.